Over the past several years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. The former entails a 30-100 fold enhancement of interleukin-2 (IL-2) production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters if it only receives a signal through the antigen-specific receptor. Our recent work has focused on the molecular mechanisms behind these two phenomena. Using IL-2 RNA accumulation and transcription reporter assays, CD28 costimulation was shown to increase the stability of IL-2 messenger(m) RNA and not to enhance the initiation of transcription. An early component of the CD28 effect was nuclear, however, as the enhancement was found in unspliced RNA and for reporter constructs containing the 3'end of the IL-2 gene. The hypothesis we are currently testing is that CD28 costimulation causes the binding or increased stability of RNA shuttle proteins that attach to IL-2 RNAs in the nucleus and accompany them out into the cytoplasm, stabilizing the RNA from degradation in both compartments. Our previous work on anergy provided evidence that the transactivation of the IL-2 gene by the AP-1 transcription factor was impaired in anergized T cell clones. We have now extended those findings with a variety of new reporter constructs to show that both the distal and proximal AP-1 response elements in the IL-2 enhancer are necessary for anergy induction. Anergy does not affect the induction of new Fos and Jun proteins which make up the AP-1 complex and has only a minor effect on the amount of complex which binds (2-fold less) and its relative affinity for the AP-1 consensus sequence (<2-fold difference). We are currently testing the hypothesis that anergy blocks the induction of AP-1 transactivating activity.